Identification of NTN4, TRA1, and STC2 as prognostic markers in breast cancer in a screen for signal sequence encoding proteins.

PURPOSE: In a previous screen using a signal-trap library, we identified a number of secreted proteins up-regulated in primary tumor cells isolated from invasive breast cancers. The purpose of this study was to assess the expression of these genes in human invasive breast tumors and to determine the significance of their expression for prognosis in breast cancer. EXPERIMENTAL DESIGN: A tissue microarray containing 245 invasive breast tumors from women treated with curative surgery followed by anthracycline-based chemotherapy and hormone therapy for the estrogen receptor (ER)-positive tumors was screened by in situ hybridization with probes against thrombospondin 3 (TSP3), insulin-like growth factor binding protein 7 (IGFBP7), tumor rejection antigen 1 (TRA1), stanniocalcin 2 (STC2), and netrin 4 (NTN4). Correlations between categorical variables were done using the chi(2) test and Fisher's exact test. Cumulative survival probabilities were calculated using the Kaplan-Meier method and multivariate survival analysis was done with Cox hazard model. A series of breast cancers were also stained with NTN4 antibodies. RESULTS: All five genes examined were expressed in invasive breast tumor cells. NTN4 protein expression was also confirmed by immunohistochemistry. Together, these data validate the design and screening of the signal-trap library. Univariate survival analysis revealed that expressions of TRA1, STC2, and NTN4 are correlated with longer disease-free survival and that TRA1 and NTN4 are associated with longer overall survival. Multivariate analysis showed that NTN4 is an independent prognostic factor of overall survival. CONCLUSIONS: This article describes the identification of three secreted proteins, NTN4, TRA1, and STC2, as potential novel prognostic markers in breast cancer.

A recombinant H1 histone-based system for efficient delivery of nucleic acids.

We describe here a unique transfer system based on a truncated form of the human linker histone H1F4 for the delivery of nucleic acids to a variety of cells. The efficiency of truncated histone H1.4F was assessed using both primary mammalian and immortalised insect and mammalian cell lines. Our results indicated that recombinant histone H1.4F was able to deliver DNA, dsRNA and siRNA in all cells tested. Quantitative analysis based on reporter gene expression or silencing of target genes revealed that the transfection efficiency of histone H1.4F was comparable to, or better than, liposome-based systems. Notably, the efficiency of histone H1.4F was associated with very low toxicity for transfected cells. The human H1.4F recombinant protein is easily purified in large-scale from bacterial lysates using inexpensive simplified processing. This versatile transfection system represents an important advance in the field of gene delivery and an improvement over earlier nucleic acid delivery methods.

A role for glial cell derived neurotrophic factor induced expression by inflammatory cytokines and RET/GFR alpha 1 receptor up-regulation in breast cancer.

By screening a tissue microarray of invasive breast tumors, we have shown that the receptor tyrosine kinase RET (REarranged during Transfection) and its coreceptor GFR alpha 1 (GDNF receptor family alpha-1) are overexpressed in a subset of estrogen receptor-positive tumors. Germ line-activating oncogenic mutations in RET allow this receptor to signal independently of GFR alpha 1 and its ligand glial cell-derived neurotrophic factor (GDNF) to promote a spectrum of endocrine neoplasias. However, it is not known whether tumor progression can also be driven by receptor overexpression and whether expression of GDNF, as has been suggested for other neurotrophic factors, is regulated in response to the inflammatory microenvironment surrounding many epithelial cancers. Here, we show that GDNF stimulation of RET(+)/GFR alpha 1(+) MCF7 breast cancer cells in vitro enhanced cell proliferation and survival, and promoted cell scattering. Moreover, in tumor xenografts, GDNF expression was found to be up-regulated on the infiltrating endogenous fibroblasts and to a lesser extent by the tumor cells themselves. Finally, the inflammatory cytokines tumor necrosis factor-alpha and interleukin-1 beta, which are involved in tumor promotion and development, were found to act synergistically to up-regulate GDNF expression in both fibroblasts and tumor cells. These data indicate that GDNF can act as an important component of the inflammatory response in breast cancers and that its effects are mediated by both paracrine and autocrine stimulation of tumor cells via signaling through the RET and GFR alpha 1 receptors.